Effect of glycerol on glucokinase activity: loss of cooperative behavior with respect to glucose

Glucose phosphorylation catalyzed by rat liver glucokinase measured at saturating concentrations of MgATP2- shows a cooperative response with respect to glucose in the concentration range 0.25-5 mM with a Hill coefficient of 1.6. In this range of glucose concentrations, the degree of cooperativity was dependent on the presence of glycerol in the assay mixture, and it decreased progressively and disappeared completely as the glycerol concentration reached about 20% (v/v) glycerol. If attention was confined to concentrations above 5 mM, no cooperativity could be detected either in the absence or in the presence of glycerol. The limiting velocity of the glucokinase reaction (measured at saturating concentrations of glucose and MgATP2-), and the half-saturation concentration for glucose and MgATP2- were all decreased by about 50-60% as the glycerol concentration was raised from zero to 30% (v/v). The presence of glycerol had no effect on the qualitative inhibition patterns of MgADP2-, glucose 6-phosphate, or N-acetylglucosamine, and only slight effects on the quantitative half-saturation values and inhibition constants. All of these effects caused by glycerol were fully reversible by decreasing the concentration of glycerol by dilution. Simulation studies based on the "mnemonical" model of glucokinase action proposed earlier [A. C. Storer and A. Cornish-Bowden (1977) Biochem. J. 165, 61-69] show that the effects of glycerol on glucokinase-catalyzed glucose phosphorylation can simply be explained assuming the glycerol favors the existence of the conformation of the enzyme with a higher affinity for glucose and thus supports the model.     

Comparison of steady-state kinetics of thiophosphorylated versus unphosphorylated smooth muscle myosin

(i) The steady-state kinetic data obtained with purified gizzard and uterus smooth muscle myosins indicated the presence of a plateau region on the substrate-saturation curves. Hill plots of these data provided evidence for mixed positive and negative cooperative interactions. In contrast, when gizzard myosin was prepared according to the method of A. Sobieszek and R.D. Bremel (1975, Eur. J. Biochem.55, 49–60), the saturation curve in the presence of CaATP was hyperbolic and no cooperativity of the binding site(s) was discerned. However, in the presence of MgATP although the curve appeared hyperbolic the Hill plot of the data was biphasic with negative cooperativity at low MgATP concentration, (ii) When thiophosphorylated gizzard myosin was used for kinetic analysis, the plateau region in the presence of MnATP was eliminated from the saturation curve and this curve became hyperbolic. However, in the presence of MgATP, although the plateau was almost eliminated, the saturation curve was still biphasic with either no or greatly reduced negative cooperativity of binding sites at low MgATP concentrations but positive cooperativity of binding at high MgATP concentrations. In addition, the thiophosphorylation of myosin also increased the K_m and V of MgATP and MnATP, thus indicating weaker affinity for these substrates with thiophosphorylated myosin. (iii) Gizzard myosin also hydrolyzed other nucleotides (the order of rates being CTP = ITP > ATP = UTP > GTP), therefore saturation kinetics using different nucleotides as substrates was also carried out. The saturation curves with each nucleotide were different i.e., hyperbolic with CTP, sigmoid with GTP, hyperbolic with biphasic Hill plot with ITP, and possessing plateau with UTP. In addition, it was observed that the kinetic pattern with each nucleotide was very sensitive to temperature and pH.     

Comparative properties of multiple forms of rabbit muscle phosphofructokinase

Some properties of three interconvertible forms of rabbit muscle phosphofructokinase specifically eluted from DEAE-cellulose with 19 mM citrate in 0.1 M tris-phosphate buffer, pH 8,0 (I), with 0,3 M buffer (II) and 1.5 M NaCl (III) are compared. Forms I-III differ in specific activities, alpha-helices content and sedimentation properties. The kinetic behaviour of forms I and III in 25 mM glycylglycine-beta-glycerophosphate, pH 8.3, at inhibitory ATP concentrations is characterized by biphasic velocity versus fructose-6-phosphate concentration curves with nH = 1.0 and 2.3, but with different V and [S]0.5 for the respective forms. At pH 6.8 from I is characterized by the kinetic curves with a lag period, while form III--by that with a burst. Form I reveals negative cooperativity in initial and stationary velocities at low substrate concentrations. The stationary velocity of form III is characterized by negative cooperativity within the whole concentration range studied. At pH 7.0 both forms are inhibited by citrate according to the initial and stationary velocities; however, the Ki values are different. The complex kinetic behaviour of phosphofructokinase corresponds to its complex chromatographic and sedimentation behaviour. The multiplicity of the enzyme forms seems to be due to a complex set of its oligomers and conformers and a hysteretic type of transitions between them as well as to its phosphorylation and possible binding of ligands.     

Effect of calixarene C-107 on kinetic parameters of Na+, K(+)-ATPase in the plasma membrane of the uterus myocytes

The inhibitory action of calixarene C-107 (5,17-diamino(2-pyridyl)methylphosphono- 11,23-di-tret-butyl-26,28-dihydroxy-25,27-dipropoxy-calix[4]arene) on Na+, K(+)-ATPase activity kinetic properties of myometrium perforated plasma membrane was investigated. It has been shown that the calixarene C-107 inhibiting Na+, K(+)-ATPase does not change the kinetic parameters (Km, nH) of reaction velocity dependence on substrate concentration. The constant Ka of enzyme activation by MgCl2 has complex dependence on calixarene C-107 concentration: it increases twice with growth of calixarene concentration up to 50 nM and decreases to the control level with further growth of calixarene concentration. The Hill cooperativity coefficient nH of activation by MgCl2 does not vary in the presence of calixarene C-107. Both ATP and MgCl2 have no influence on Na+, K(+)-ATPase constant of inhibition by calixarene C-107, but an increase of concentration of the mentioned physiological compounds causes the growth of cooperativity coefficient nH of enzymatic reaction inhibition by calixaren C-107.     

On the temperature- and salt-dependent conformation change in human erythrocyte pyruvate kinase

The influence of temperature, K+, Mg2+ and fructose 1,6-bisphosphate on human red cell pyruvate kinase was investigated. Kinetic measurements between 4 degrees C and 43 degrees C revealed a remarkable influence of the temperature on the allosteric behaviour of the enzyme. Below a transition region between 15 degrees C and 20 degrees C (as obtained from an Arrhenius plot) the enzyme shows non-cooperative behaviour, as can be deduced from Michaelis-Menten, Hill and Scatchard plots. At temperatures above 20 degrees C cooperativity increases with rising temperature. This effect becomes even more pronounced at higher temperatures upon addition of increasing amounts of K+ and Mg2+ accompanied by a slight decrease of the reaction velocity. Fructose 1,6-bisphosphate, however, abolishes cooperativity at every temperature and salt concentration measured. Difficulties which arise in evaluating the correct values of V, Km and the Hill coefficient nH with cooperative systems are met by using a computer program of Wieker, Johannes and Hess, especially designed for the determination of kinetic parameters obtained from sigmoidal steady-state kinetics.     

Effect of digitonin on the activity of Na,K-ATPase from bovine brain

The kinetic properties of intact and digitonin-treated Na,K-ATPase from bovine brain were studied. The temperature dependence curve for the rate of ATP hydrolysis under optimal conditions (upsilon 0) in the Arrhenius plots shows a break at 19-20 degrees. The temperature dependence curves for Km' and Km" have breaks at the same temperatures, while the Arrhenius plot for V is linear. The value of the Hill coefficient (nH) for ATP at 37 degrees is variable depending on ATP concentration, i. e. it is less than 1 at ATP concentrations below 50 mkM and is increased up to 3.2 at higher concentrations of the substrate. At high ATP concentrations the value of nH depends on temperature, falling down to 2.1 at 23 degrees and then down to 1 within the temperature range of 21-19 degrees. A further decrease in temperature does not significantly affect the nH value. Digitonin irreversibly inhibits Na, K-ATPase. ATP hydrolysis is more sensitive to the effect of the detergent than is nNPP hydrolysis, i. e. after complete inhibition of the ATPase about 40% of the phosphatase activity are retained. Treatment of Na,K-ATPase by digitonin results in elimination of the breaks in the Arrhenius plots for upsilon 0, Km' and Km", whereas the temperature dependence plot of V remains linear. Simultaneously digitonin eliminates the positive cooperativity of the enzyme for ATP. It is assumed that Na, K-ATPase from bovine brain is an oligomer of the (alpha beta) 4 type. Digitonin changes the type of interaction between the protomers within the oligomeric complex by changing the lipid environment of the enzyme or the type of protein -- lipid interactions.     

Kinetic evidence for a ''mnemonical'' mechanism for rat liver glucokinase.

Inhibition studies of glucokinase were carried out with the products of the reaction, glucose 6-phosphate and MgADP-, as well as with ADP3-, Mg2+ and ATP4-. The results of these, together with those of kinetic studies of the uninhibited reaction described previously [Storer & Cornish-Bowden (1976) Biochem. J. 159, 7-14], indicate that the enzyme obeys a 'mnemonical' mechanism. This implies that the co-operativity observed with glucose as substrate arises because glucose binds differentially to two forms of the free enzyme that are not in equilibrium under steady-state conditions. The mechanism predicts the decrease in glucose co-operativity observed at low concentrations of MgATP2-. The product-inhibition results suggest that glucose 6-phosphate is released first and that it is possibly displaced by MgATP2- in a concerted reaction.     

Temperature effects on the allosteric transition of ATP sulfurylase from Penicillium chrysogenum

The effects of temperature on the initial velocity kinetics of allosteric ATP sulfurylase from Penicillium chrysogenum were measured. The experiments were prompted by the structural similarity between the C-terminal regulatory domain of fungal ATP sulfurylase and fungal APS kinase, a homodimer that undergoes a temperature-dependent, reversible dissociation of subunits over a narrow temperature range. Wild-type ATP sulfurylase yielded hyperbolic velocity curves between 18 and 30 degrees C. Increasing the assay temperature above 30 degrees C at a constant pH of 8.0 increased the cooperativity of the velocity curves. Hill coefficients (n(H)) up to 1.8 were observed at 42 degrees C. The bireactant kinetics at 42 degrees C were the same as those observed at 30 degrees C in the presence of PAPS, the allosteric inhibitor. In contrast, yeast ATP sulfurylase yielded hyperbolic plots at 42 degrees C. The P. chrysogenum mutant enzyme, C509S, which is intrinsically cooperative (n(H) = 1.8) at 30 degrees C, became more cooperative as the temperature was increased yielding n(H) values up to 2.9 at 42 degrees C. As the temperature was decreased, the cooperativity of C509S decreased; n(H) was 1.0 at 18 degrees C. The cumulative results indicate that increasing the temperature increases the allosteric constant, L, i.e., promotes a shift in the base-level distribution of enzyme molecules from the high MgATP affinity R state toward the low MgATP affinity T state. As a result, the enzyme displays a true "temperature optimum" at subsaturating MgATP. The reversible temperature-dependent transitions of fungal ATP sulfurylase and APS kinase may play a role in energy conservation at high temperatures where the organism can survive but not grow optimally.     

The "regulatory" sulfhydryl group of Penicillium chrysogenum ATP sulfurylase. Cooperative ligand binding after SH modification; chemical and thermodynamic properties

ATP sulfurylase from Penicillium chrysogenum is a homohexamer that contains three free sulfhydryl groups/subunit, only one of which (designated SH-1) can be modified by disulfide, maleimide, and halide reagents under nondenaturing conditions. Modification of SH-1 has only a small effect on kcat but causes the [S]0.5 values for MgATP and SO4(2-) (or MoO4(2-) to increase by an order of magnitude. Additionally, the velocity curves become sigmoidal with a Hill coefficient (nH) of about 2 (Renosto, F., Martin, R. L., and Segel, I. H. (1987) J. Biol. Chem. 262, 16279-16288). Direct equilibrium binding measurements confirmed that [32P]MgATP binds to the SH-modified enzyme in a positively cooperative fashion (nH = 2.0) if a sulfate subsite ligand (e.g. FSO3-) is also present. [35S]Adenosine 5'-phosphosulfate (APS) binding to the SH-modified enzyme displayed positive cooperativity (nH = 1.9) in the absence of a PPi subsite ligand. The results indicate that positive cooperativity requires occupancy of the adenylyl and sulfate (but not the pyrophosphate) subsites. [35S]APS binding to the native enzyme displayed negative cooperativity (or binding to at least two classes of sites). Isotope trapping profiles for the single turnover of [35S]APS: (a) confirmed the equilibrium binding curves, (b) indicated that all six sites/hexamer are catalytically active, and (c) showed that APS does not dissociate at a significant rate from E.APS.PPi. The MgPPi concentration dependence of [35S]APS trapping was indicative of MgPPi binding to two classes of sites on both the native and SH-modified enzyme. Inactivation of the native or SH-modified enzyme by phenylglyoxal in the presence of saturating APS was biphasic. The semilog plots suggested that only half of the sites were highly protected. The cumulative data suggest a model in which pairs of sites or subunits can exist in three different states designated HH (both sites have a high APS affinity, as in the native free enzyme), LL (both sites have a low APS affinity as in the SH-modified enzyme), and LH (as in the APS-occupied native or SH-modified enzyme). Thus, the HH----LH transition displays negative cooperativity for APS binding while the LL----LH transition displays positive cooperativity. The relative reactivities of like-paired SH-reactive reagents were in the order: N-phenylmaleimide greater than N-ethylmaleimide; dithionitropyridine greater than dithionitrobenzoate; thiolyte-MQ greater than thiolyte-MB. The log kmod versus pH curve indicates that the pKa of SH-1 is greater than 9.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effect of monovalent and divalent cations on the activity of Streptococcus lactis C10 pyruvate kinase.

The pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from Streptococcus lactis C10 had an obligatory requirement for both a monovalent cation and divalent cation. NH+4 and K+ activated the enzyme in a sigmoidal manner (nH =1.55) at similar concentrations, whereas Na+ and Li+ could only weakly activate the enzyme. Of eight divalent cations studied, only three (Co2+, Mg2+ and Mn2+) activated the enzyme. The remaining five divalent cations (Cu2+, Zn2+, Ca2+, Ni2+ and Ba2+) inhibited the Mg2+ activated enzyme to varying degrees. (Cu2+ completely inhibited activity at 0.1 mM while Ba2+, the least potent inhibitor, caused 50% inhibition at 3.2 mM). In the presence of 1 mM fructose 1,6-diphosphate (Fru-1,6-P2) the enzyme showed a different kinetic response to each of the three activating divalent cations. For Co2+, Mn2+ and Mg2+ the Hill interaction coefficients (nH) were 1.6, 1.7 and 2.3 respectively and the respective divalent cation concentrations required for 50% maximum activity were 0.9, 0.46 and 0.9 mM. Only with Mn2+ as the divalent cation was there significatn activity in the absence of Fru-1,6-P2. When Mn2+ replaced Mg2+, the Fru-1,6-P2 activation changed from sigmoidal (nH = 2.0) to hyperbolic (nH = 1.0) kinetics and the Fru-1,6-P2 concentration required for 50% maximum activity decreased from 0.35 to 0.015 mM. The cooperativity of phosphoenolpyruvate binding increased (nH 1.2 to 1.8) and the value of the phosphoenolpyruvate concentration giving half maximal velocity decreased (0.18 to 0.015 mM phosphoenolyruvate) when Mg2+ was replaced by Mn2+ in the presence of 1 mM Fru-1,6-P2. The kinetic response to ADP was not altered significantly when Mn2+ was substituted for Mg2+. The effects of pH on the binding of phosphoenolpyruvate and Fru-1,6-P2 were different depending on whether Mg2+ or Mn2+ was the divalent cation.     

Reversible shift between two states of Ca2+-ATPase in human erythrocytes mediated by Ca2+ and a membrane-bound activator.

The (Ca2+ + Mg2+)-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) from human erythrocytes occurred in two different states, A-state and B-state, depending on the membrane preparation. The A-state showed low maximum activity (V) and the Ca2+ activation was characterized by a Hill coefficient, nH, of about 1 and a Michaelis constant, KCa, about 30 micron. The B-state showed high V, a nH above 1, which indicates positive cooperativity of Ca2+ activation, and KCa of about 1 micron. With varying ATP concentrations, both the A-state and B-state showed negative cooperativity and slightly different values of Km. The B-state was shifted to A-state when the membranes were exposed to low Ca2+ concentration. The shift reached 50% at approx. 0.5 micron Ca2+. At the low Ca2+ concentrations an activator was released from the membranes. The A-state was shifted to the B-state when the membranes were exposed to Ca2+ in the presence of the activator. The shift reached 50% at about 30 micron Ca2+. The recovery of high V was time dependent and lasted several minutes. Increasing concentrations of Ca2+ and activator accelerated the recovery. It is suggested that the A-state and the B-state correspond to enzyme free of activator and enzyme associated with activator, respectively. Furthermore, the two states may respresent a resting and an active state, respectively, of the calcium pump.     

Kinetic cooperativity in the concerted model for allosteric enzymes.

The cooperativity of enzyme-substrate interactions is investigated in the concerted allosteric model of Monod, Wyman and Changeux. The general case of K-V systems is considered, in which the two protomer conformational states R and T postulated in the theory differ in catalytic and binding properties. An expression for the Hill coefficient nH defined with respect to the asymptotic velocity V infinity to is analyzed in conditions which exclude substrate inhibition. Kinetic cooperativity is always positive (nH greater than 1) in the case of a dimer enzyme, and in the case of an inactive T state. Slight kinetic negative cooperativity (nH less than 1) occurs under restrictive conditions for larger numbers of protomers when the substrate binds significantly to the less active state of the enzyme, but the phenomenon remains negligible for trimers and tetramers. These conclusions differ from those obtained [A. Goldbeter, J. Mol.Biol.90 (1974) 185] with the Hill coefficient based on the absolute maximum velocity, which may exceed the experimental value V infinity to in K-V systems. The results extend those of Paulus and DeRiel [J. Mol. Biol. 97 (1975) 667] and support the view that in most cases, negative cooperativity is not compatible with a mechanism based on a concerted and conservative allosteric transition. The Hill coefficients for binding and catalysis are compared in K-V systems.     

Steady-state kinetics of smooth muscle myosin.

The kinetic properties of purified smooth muscle myosin, free of actin, have been examined. Analysis of the steady-state kinetic data revealed an intermediary plateau region on the substrate saturation curves. In addition, these data, when analyzed by Hill and Lineweaver and Burk plots, indicate both positive and negative cooperativity, suggesting at least four substrate binding sites. The plateau region was abolished when the kinetic measurements were made at pH 5.5 and 9.0. Both positive and negative cooperative effects were absent at pH 9.0 and hyperbolic kinetics was observed. In contrast, at pH 5.5, although the plateau region was abolished, the enzyme exhibited positive cooperativity of substrate binding. When either heated or urea treated enzyme was used for kinetic measurements: (i) the plateau region shifted toward higher substrate concentration range; (ii) the cooperativity of binding sites was lost at low substrate concentrations but was instead seen at higher concentrations; and (iii) the Vmax was doubled. These data have been interpreted as due to ligand-induced conformational changes in the enzyme according to J. Teipel and D. E. Koshland, Jr. (1969).     

Kinetic properties of the purified Ca2+-translocating ATPase from human erythrocyte plasma membrane

The basic kinetic properties of the solubilized and purified Ca2+-translocating ATPase from human erythrocyte membranes were studied. A complex interaction between the major ligands (i.e., Ca2+, Mg2+, H+, calmodulin and ATP) and the enzyme was found. The apparent affinity of the enzyme for Ca2+ was inversely proportional to the concentration of free Mg2+ and H+, both in the presence or absence of calmodulin. In addition, the apparent affinity of the enzyme for Ca2+ was significantly increased by the presence of calmodulin at high concentrations of MgCl2 (5 mM), while it was hardly affected at low concentrations of MgCl2 (2 mM or less). In addition, the ATPase activity was inhibited by free Mg2+ in the millimolar concentration range. Evidence for a high degree of positive cooperativity for Ca2+ activation of the enzyme (Hill coefficient near to 4) was found in the presence of calmodulin in the slightly alkaline pH range. The degree of cooperativity induced by Ca2+ in the presence of calmodulin was decreased strongly as the pH decreased to acid values (Hill coefficient below 2). In the absence of calmodulin, the Hill coefficient was 2 or slightly below over the whole pH range tested. Two binding affinities of the enzyme for ATP were found. The apparent affinity of the enzyme for calmodulin was around 6 nM and independent of the Mg2+ concentration. The degree of stimulation of the ATPase activity by calmodulin was dependent on the concentrations of both Ca2+ and Mg2+ in the assay system.     

Mechanistic origin of the kinetic cooperativity of hexokinase type L1 from wheat germ

A theoretical analysis is presented which shows that initial velocity data for hexokinase L1 catalysis of glucose phosphorylation by MgATP cannot be reconciled with the observed rate of the 'mnemonical' conformational transition which has been proposed to account for the kinetic cooperativity of the enzyme. The basic kinetic properties of hexokinase L1 and other allegedly 'mnemonical' enzymes appear to be fully consistent with an ordered ternary-complex mechanism in which the leading substrate participates in abortive-complex formation. It is concluded that, so far, no enzyme displaying kinetic cooperativity has been convincingly demonstrated to operate by a 'mnemonical' type of reaction mechanism.     

Kinetics of the acid pump in the stomach. Proton transport and hydrolysis of ATP and p-nitrophenyl phosphate by the gastric H,K-ATPase

Hydrolysis of adenosine 5'-triphosphate (ATP) and p-nitrophenyl phosphate by the hydrogen ion-transporting potassium-stimulated adenosine triphosphatase (H,K-ATPase) was investigated. Hydrolysis of ATP was studied at pH 7.4 in vesicles treated with the ionophore nigericin. The kinetic analysis showed negative cooperativity with one high affinity (Km1 = 3 microM) and one low affinity (Km2 = 208 microM) site for ATP. The rate of hydrolysis decreased at 2000 microM ATP indicating a third site for ATP. When the pH was decreased to 6.5 the experimental results followed Michaelis-Menten enzyme kinetics with one low affinity site (Km = 116 microM). Higher concentrations than 750 microM ATP were inhibitory. Proton transport was measured as accumulation of acridine orange in vesicles equilibrated with 150 mM KCl. The transport at various concentrations of ATP in the pH interval from 6.0 to 8.0 correlated well with the Hill equation with a Hill coefficient between 1.5-1.9. The concentration of ATP resulting in half-maximal transport rate (S0.5) increased from 5 microM at pH 6.0 to 420 microM at pH 8.0. At acidic pH the rate of proton transport decreased at 1000 microM ATP. The K+-stimulated p-nitrophenylphosphatase (pNPPase) activity resulted in a Hill coefficient close to 2 indicating cooperative binding of substrate. The pNPPase was noncompetitively inhibited by ATP and ADP; half-maximal inhibition was obtained at 2 and 100 microM, respectively. Phospholipase C-treated vesicles lost 80% of the pNPPase activity, but the Hill coefficient did not change. These kinetic results are used for a further development of the reaction scheme of the H,K-ATPase.     

Studies of the phosphoenzyme intermediate of the yeast plasma membrane proton-translocating ATPase

The yeast plasma membrane proton-pumping ATPase forms a phosphorylated intermediate during the hydrolysis of ATP. The fraction of enzyme phosphorylated during steady-state ATP hydrolysis was studied as a function of substrate concentration (MgATP), Mg2+ concentration, and pH. The dependence of the fraction of enzyme phosphorylated on the concentration of MgATP is sigmoidal, and the isotherms can be fit with parameters and mechanisms similar to those used to describe ATP hydrolysis. The isotherm is significantly more sigmoidal at pH 5.5 than at pH 6.0, with the limiting percentage (100.mol of phosphate/mol of enzyme) of enzyme phosphorylated being 70% and 6%, respectively, at the two pH values. The maxima in the steady-state rate of ATP hydrolysis occur at higher concentrations of Mg2+ and higher pH than the maxima in the fraction of enzyme phosphorylated. This suggests that the rate-determining step for ATP hydrolysis is different from that for enzyme phosphorylation and the hydrolysis of phosphoenzyme is enhanced by Mg2+ and high pH. The rate of phosphoenzyme formation was investigated with the quenched-flow method, but only a lower bound of 140 s-1 could be obtained for the rate constant at MgATP concentrations greater than 2.5 mM. Since the turnover number for ATP hydrolysis under similar conditions is 14 s-1, the rate-determining step in ATP hydrolysis occurs after enzyme phosphorylation.     

ATP sulfurylase from Penicillium chrysogenum. Molecular basis of the sigmoidal velocity curves induced by sulfhydryl group modification

ATP sulfurylase from Penicillium chrysogenum is a noncooperative homooligomer containing three free sulfhydryl groups per subunit. Under nondenaturing conditions, one SH group per subunit was modified by 5,5'-dithiobis-(2-nitrobenzoate), or N-ethylmaleimide. Modification had only a small effect on kcat, but markedly increased the [S]0.5 values for the substrates, MgATP and SO4(2-). MgATP and adenosine-5'-phosphosulfate protected against modification. The SH-modified enzyme displayed sigmoidal velocity curves for both substrates with Hill coefficients (nH) of 2. Fluorosulfonate (FSO3-) and other dead-end inhibitors competitive with SO4(2-) activated the SH-modified enzyme at low SO4(2-) concentration. In order to determine whether the sigmoidicity resulted from true cooperative binding (as opposed to a kinetically based mechanism), the shapes of the binding curves were established from the degree of protection provided by a ligand against phenylglyoxal-dependent irreversible inactivation under noncatalytic conditions. Under standard conditions (0.05 M Na-N-(2-hydroxyethyl)piperazine-N'-3-propanesulfonic acid buffer, pH 8, 30 degrees C, and 3mM phenylglyoxal) the native enzyme was inactivated with a k of 2.67 +/- 0.25 X 10-3 s-1, whereas k for the SH-modified enzyme was 5.44 +/- 0.27 X 10-3 s-1. The increased sensitivity of the modified enzyme resulted from increased reactivity of ligand-protectable groups. Both the native and the SH-modified enzyme displayed hyperbolic plots of delta k (i.e. protection) versus [MgATP], or [FSO3-], or [S2O3(2-]) in the absence of coligand (nH = 0.98 +/- 0.06). The plots of delta k versus [ligand] for the native enzyme were also hyperbolic in the presence of a fixed concentration of coligand. However, in the presence of a fixed [FSO3-] or [S2O3(2-]), the delta k versus [MgATP] plot for the SH-modified enzyme was sigmoidal, as was the plot of delta k versus [FSO3-] or [S2O3(2-]) in the presence of a fixed [MgATP]. The nH values were 1.92 +/- 0.09. The results indicate that substrates (or analogs) bind hyperbolically to unoccupied SH-modified subunits, but in a subunit-cooperative fashion to form a ternary complex.     

Allosteric properties, substrate specificity, and subsite affinities of honeybee alpha-glucosidase I

The substrate specificity of honeybee alpha-glucosidase I, a monomeric enzyme was kinetically investigated. Unusual kinetic features were observed in the cleavage reactions of sucrose, maltose, p-nitrophenyl alpha-glucoside, phenyl alpha-glucoside, turanose, and maltodextrin (DP = 13). At relatively high substrate concentrations, the velocities of liberation of fructose from sucrose, glucose from maltose, p-nitrophenol from p-nitrophenyl alpha-glucoside, and phenol from phenyl alpha-glucoside were accelerated, and so the Lineweaver-Burk plots were convex, indicating negative kinetic cooperativity: the Hill coefficients were calculated to be 0.50, 0.64, 0.50, and 0.67 for sucrose, maltose, p-nitrophenyl alpha-glucoside, and phenyl alpha-glucoside, respectively. For the degradation of turanose and maltodextrin, the enzyme showed a sigmoidal curve in v versus s plots and thus catalyzed the reaction with positive kinetic cooperativity. The Lineweaver-Burk plots were concave and the Hill coefficients were 1.2 and 1.5 for turanose and maltodextrin, respectively. These unique properties cannot be interpreted by the reaction mechanism that Huber and Thompson proposed: (1973) Biochemistry 12, 4011-4020. The rate parameters for the hydrolysis of sucrose, maltose, p-nitrophenyl alpha-glucoside and phenyl alpha-glucoside were estimated by extrapolating the linear part of the Lineweaver-Burk plots at low substrate concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

The control of adenylate cyclase by calcium in turkey erythrocyte ghosts.

The adenylate cyclase of turkey erythrocytes is inhibited by low concentrations of calcium. Calcium binds to the enzyme system so tightly that the enzyme can compete with ethylene glycol bis(beta-aminoethyl ether)-N, N1-tetraacetic acid (EGTA) for the metal. The calcium binding site is shown to be distinct from the magnesium binding sites required for activity. Thus Ca2+ functions as a negative allosteric effector. Calcium decreases dramatically the V max of the catecholamine-stimulated activity without affecting the affinity for the hormone or for the substrate ATP. The cooperativity in the response toward Mg2+ dependence (Hill coefficient, nH equals 3) is also unaffected by Ca2+ where as the S0.5 (concentration yielding one-half V max) for Mg2+ is affected only slightly. The Ca2+ effect is cooperative (nH equals 2) and therefore brought about by a cluster of Ca2+ binding sites. Mn2+ can substitute for Mg2+ as the enzyme activator but the Mn2+-activated enzyme is no longer inhibited by Ca2+. The possible physiological significance of the Ca2+ effect is discussed.